Treating bladder cancer patients and identifying bladder cancer patients responsive to treatment

ABSTRACT

This documents provided methods and materials involved in treating bladder cancer. This document also provides methods and materials involved in identifying bladder cancer patients likely or unlikely to respond to treatment (e.g., BCG therapy).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. 371 ofInternational Application No. PCT/US2012/066370, having an InternationalFiling Date of Nov. 21, 2012, which claims the benefit of U.S.Provisional Application Ser. No. 61/563,426, filed Nov. 23, 2011. Thedisclosure of the prior application is considered part of (and isincorporated by reference in) the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treatingbladder cancer patients. This document also relates to methods andmaterials involved in identifying bladder cancer patients likely orunlikely to respond to treatment (e.g., Bacillus Calmette-Guerin (BCG)therapy).

2. Background Information

Bladder cancer is a significant health problem in the United States,with an estimated 70,530 new cases diagnosed in 2010 and 14,680estimated deaths. Bladder cancer currently ranks fourth in incidenceamong all cancers in males and eleventh among cancers in women. Inrecent decades, the overall incidence of bladder cancer appears to berising, particularly among patients more than 55 years of age.

Transitional cell carcinoma, the most common pathologic subtype ofbladder cancer, is observed in over 90% of tumors. TheTumor-Node-Metastases (TNM) classification (American Joint Committee onCancer) is a commonly used and accepted staging system for bladdercancer. In this system, bladder cancer tumors have specifically definedcharacteristics. In particular, non-muscle invasive tumors, includingpapillary tumors confined to the epithelial mucosa, are termed Tatumors. In contrast, tumors invading the subepithelial tissue (i.e.,lamina propria) are defined as T1 tumors. Tumors with a distinctmorphology and a dynamic phenotype are known as carcinoma in situ (Tis).Invasive tumors (T2-4a and T2-4b) are further defined and divided on thebasis and extent of their invasive character following histopathologicevaluation.

SUMMARY

This document provides methods and materials involved in treatingbladder cancer. This document also provides methods and materialsinvolved in identifying bladder cancer patients likely or unlikely torespond to treatment (e.g., BCG therapy). For example, this documentprovides methods and materials involve in using the ratio oftumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1 lymphoidcells to identify bladder cancer patients (e.g., Tis bladder cancerpatients) who are likely to respond to an anti-cancer immunotherapy(e.g., BCG therapy). As described herein, patients having an initialbladder tissue biopsy with more tumor-infiltrating Th2 lymphoid cellsthan tumor-infiltrating Th1 lymphoid cells can be identified orclassified as being likely to respond to BCG therapy, while patientshaving an initial bladder tissue biopsy that does not have moretumor-infiltrating Th2 lymphoid cells than tumor-infiltrating Th1lymphoid cells can be identified or classified as being unlikely torespond to BCG therapy.

This document also provides methods and materials involve in using thelevel of eosinophil infiltration and/or degranulation to identifybladder cancer patients (e.g., Tis bladder cancer patients) who arelikely or unlikely to respond to an anti-cancer immunotherapy (e.g., BCGtherapy). As described herein, patients having an initial bladder tissuebiopsy with elevated eosinophil infiltration and/or degranulation can beidentified or classified as being likely to respond to BCG therapy,while patients having an initial bladder tissue biopsy that lackselevated eosinophil infiltration and/or degranulation can be identifiedor classified as being unlikely to respond to BCG therapy. In somecases, both the ratio of tumor-infiltrating Th2 lymphoid cells totumor-infiltrating Th1 lymphoid cells and the level of eosinophilinfiltration and/or degranulation can be used in combination to identifybladder cancer patients (e.g., Tis bladder cancer patients) who arelikely to respond to an anti-cancer immunotherapy (e.g., BCG therapy).

Having the ability to identify bladder cancer patients based, at leastin part, on the ratio of tumor-infiltrating Th2 lymphoid cells totumor-infiltrating Th1 lymphoid cells and/or the level of eosinophilinfiltration and/or degranulation using a biopsy sample (e.g., aninitial biopsy sample) can allow clinicians and patients to selectappropriate treatment options with an increased degree of certainty.

In general, one aspect of this document features a method for assessingthe likelihood of a bladder cancer patient to respond to an anti-cancerimmunotherapy. The method comprises, or consists essentially of,determining whether or not the patient contains bladder cancer tissuewith a ratio of tumor-infiltrating Th2 lymphoid cells totumor-infiltrating Th1 lymphoid cells indicative of an increasedlikelihood of responding to the anti-cancer immunotherapy. The patientcan be a human patient. The method can comprise determining whether ornot a bladder tissue biopsy sample from the patient contains the bladdercancer tissue with the ratio. The ratio can be a ratio of GATA-3⁺ toT-bet⁺ tumor-infiltrating T cells. The method can comprise determiningwhether or not the patient contains bladder cancer tissue with a levelof eosinophil infiltration or degranulation indicative of an increasedlikelihood of responding to the anti-cancer immunotherapy. Theanti-cancer immunotherapy can be BCG therapy.

In another aspect, this document features a method for assessing thelikelihood of a bladder cancer patient to respond to an anti-cancerimmunotherapy. The method comprises, or consists essentially of, (a)determining whether or not the patient contains bladder cancer tissuewith a ratio of tumor-infiltrating Th2 lymphoid cells totumor-infiltrating Th1 lymphoid cells indicative of an increasedlikelihood of responding to the anti-cancer immunotherapy, (b)classifying the patient as being likely to respond to an anti-cancerimmunotherapy if the patient contains the bladder cancer tissue with theratio, and (c) classifying the patient as being unlikely to respond toan anti-cancer immunotherapy if the patient does not contain the bladdercancer tissue with the ratio. The patient can be a human patient. Themethod can comprise determining whether or not a bladder tissue biopsysample from the patient contains the bladder cancer tissue with theratio. The ratio can be a ratio of GATA-3⁺ to T-bet⁺ tumor-infiltratingT cells. The method can comprise determining whether or not the patientcontains bladder cancer tissue with a level of eosinophil infiltrationor degranulation indicative of an increased likelihood of responding tothe anti-cancer immunotherapy. The anti-cancer immunotherapy can be BCGtherapy.

In another aspect, this document features a method for assessing thelikelihood of a bladder cancer patient to respond to an anti-cancerimmunotherapy, wherein the method comprises, or consists essentially of,(a) detecting the presence of a ratio of bladder tumor-infiltrating Th2lymphoid cells to bladder tumor-infiltrating Th1 lymphoid cellsindicative of an increased likelihood of responding to the anti-cancerimmunotherapy, and (b) classifying the patient as being likely torespond to the anti-cancer immunotherapy. The patient can be a humanpatient. The ratio can be a ratio of GATA-3⁺ to T-bet⁺ bladdertumor-infiltrating T cells. The method can comprise detecting thepresence of a level of eosinophil infiltration or degranulationindicative of an increased likelihood of responding to the anti-cancerimmunotherapy. The anti-cancer immunotherapy can be BCG therapy.

In another aspect, this document features a method for assessing thelikelihood of a bladder cancer patient to respond to an anti-cancerimmunotherapy. The method comprises, or consists essentially of,determining whether or not the patient contains bladder cancer tissuewith a level of eosinophil infiltration or degranulation indicative ofan increased likelihood of responding to the anti-cancer immunotherapy.

In another aspect, this document features a method for assessing thelikelihood of a bladder cancer patient to respond to an anti-cancerimmunotherapy. The method comprises, or consists essentially of, (a)determining whether or not the patient contains bladder cancer tissuewith a level of eosinophil infiltration or degranulation indicative ofan increased likelihood of responding to the anti-cancer immunotherapy,(b) classifying the patient as being likely to respond to an anti-cancerimmunotherapy if the patient contains the bladder cancer tissue with thelevel, and (c) classifying the patient as being unlikely to respond toan anti-cancer immunotherapy if the patient does not contain the bladdercancer tissue with the level.

In another aspect, this document features a method for assessing thelikelihood of a bladder cancer patient to respond to an anti-cancerimmunotherapy. The method comprises, or consists essentially of, (a)detecting the presence of a level of eosinophil infiltration ordegranulation indicative of an increased likelihood of responding to theanti-cancer immunotherapy, and (b) classifying the patient as beinglikely to respond to the anti-cancer immunotherapy.

In another aspect, this document features a method for treating bladdercancer. The method comprises, or consists essentially of, (a) detectingthe presence, in bladder cancer tissue of a bladder cancer patient, of aratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1lymphoid cells indicative of a decreased likelihood of responding to theanti-cancer immunotherapy, and (b) performing radical cystectomyprocedure on the patient to treat the bladder cancer. The patient can bea human patient. The ratio can be a ratio of GATA-3⁺ to T-bet⁺tumor-infiltrating T cells. The method can comprise detecting thepresence, in bladder cancer tissue of a bladder cancer patient, of alevel of eosinophil infiltration or degranulation indicative of adecreased likelihood of responding to the anti-cancer immunotherapy. Theanti-cancer immunotherapy can be BCG therapy. The patient can receivethe radical cystectomy procedure without having received a previous BCGtherapy.

In another aspect, this document features a method for treating bladdercancer, The method comprises, or consists essentially of, (a) detectingthe presence, in bladder cancer tissue of a bladder cancer patient, of aratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1lymphoid cells indicative of a decreased likelihood of responding to theanti-cancer immunotherapy, and (b) administering intravesical mitomycinC or thiotepa chemotherapy to the patient to treat the bladder cancer.The patient can be a human patient. The ratio can be a ratio of GATA-3⁺to T-bet⁺ tumor-infiltrating T cells. The method can comprise detectingthe presence, in bladder cancer tissue of a bladder cancer patient, of alevel of eosinophil infiltration or degranulation indicative of adecreased likelihood of responding to the anti-cancer immunotherapy. Theanti-cancer immunotherapy can be BCG therapy. The intravesical mitomycinC or thiotepa chemotherapy can be administered to the patient before thepatient receives BCG therapy.

In another aspect, this document features a method for treating bladdercancer. The method comprises, or consists essentially of, (a) detectingthe presence, in bladder cancer tissue of a bladder cancer patient, of alevel of eosinophil infiltration or degranulation indicative of adecreased likelihood of responding to the anti-cancer immunotherapy, and(b) performing radical cystectomy procedure on the patient to treat thebladder cancer. The patient can be a human patient. The ratio can be aratio of GATA-3⁺ to T-bet⁺ tumor-infiltrating T cells. The method cancomprise detecting the presence, in bladder cancer tissue of a bladdercancer patient, of a ratio of tumor-infiltrating Th2 lymphoid cells totumor-infiltrating Th1 lymphoid cells indicative of a decreasedlikelihood of responding to the anti-cancer immunotherapy. Theanti-cancer immunotherapy can be BCG therapy. The patient can receivethe radical cystectomy procedure without having received a previous BCGtherapy.

In another aspect, this document features a method for treating bladdercancer. The method comprises, or consists essentially of, (a) detectingthe presence, in bladder cancer tissue of a bladder cancer patient, of alevel of eosinophil infiltration or degranulation indicative of adecreased likelihood of responding to the anti-cancer immunotherapy, and(b) administering intravesical mitomycin C or thiotepa chemotherapy tothe patient to treat the bladder cancer. The patient can be a humanpatient. The ratio can be a ratio of GATA-3⁺ to T-bet⁺tumor-infiltrating T cells. The method can comprise detecting thepresence, in bladder cancer tissue of a bladder cancer patient, of aratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1lymphoid cells indicative of a decreased likelihood of responding to theanti-cancer immunotherapy. The anti-cancer immunotherapy can be BCGtherapy. The intravesical mitomycin C or thiotepa chemotherapy can beadministered to the patient before the patient receives BCG therapy.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1. T cell subtype-specific antibodies identify unique T cellpopulations infiltrating Tis bladder tumors Immunohistochemistry usingantibodies specific for the master transcription factors, GATA-3 andT-bet, identify infiltrating Th2 polarized (brown staining cells) andTh1 polarized (red staining cells) lymphocytes, respectively. Scalebar=50 μm.

FIG. 2 is a graph plotting the relative number of bladder tumorinfiltrating GATA-3⁺ (G) vs. T-bet⁺ (T) T cells (G/T ratio) at the timeof initial biopsy/diagnosis. The G/T ratio differentiates Tis bladdercancer patients who are responsive from those who are un-responsive(refractory) to BCG immune therapy. G/T was scored for individualpatient biopsies (n=9-10 per group) following immunohistochemicalstaining with antibodies specific for the respective transcriptionfactors.

FIG. 3 contains photographs of Tis bladder tumors stained with H&E or aneosinophil peroxidase-specific monoclonal antibody (EPX-mAb).Significant levels of eosinophil infiltration accompanied by activation(i.e., eosinophil degranulation) were observed Immunohistochemistryusing EPX-mAb demonstrates a robust tissue eosinophilia in Tis bladdertumors (red staining cells) that is often accompanied by eosinophilactivation leading to areas of eosinophil peroxidase release/deposition(i.e., red staining areas of extracellular matrix). Scale bar=100 μm.

FIG. 4 is a graph plotting an the number of eosinophils or degranulatingeosinophils present within tissue (EAI) for biopsies obtained at thetime of initial diagnosis from Tis bladder cancer patients who wereresponsive or un-responsive (refractory) to BCG immune therapy. Biopsiesfrom normal patients were used as a control. The EAI differentiatedpatients who are responsive from those who are un-responsive(refractory) to BCG immune therapy. EAI was scored for individualpatient biopsies (n=9-10 per group) following immunohistochemicalstaining with the EPX-mAb.

FIG. 5 is a graph plotting a Th2 biomarker signature for Tis bladdertumors from patients who were responsive or un-responsive (refractory)to BCG immune therapy. The Th2 biomarker signature represents a robustmetric or score that stratifies bladder cancer patients into subgroupseither responsive or refractory to BCG immune therapy. The numericalproduct of the signature derived from evaluations of the G/T ratio andEAI of a given bladder tumor (i.e., Th2 biomarker signature score)provided a quantifiable measure that was predictive of patient outcomefollowing BCG immune therapy.

FIG. 6 is a flow chart plotting possible bladder cancer treatments forbladder cancer patients identified as being likely or unlikely torespond to anti-cancer immunotherapy (e.g., BCG therapy).

FIG. 7 is a flow chart plotting possible bladder cancer treatments forbladder cancer patients identified as being likely or unlikely torespond to anti-cancer immunotherapy (e.g., BCG therapy).

DETAILED DESCRIPTION

This document provides methods and materials involved in treatingbladder cancer. This document also provides methods and materialsinvolved in identifying bladder cancer patients likely or unlikely torespond to treatment (e.g., BCG therapy). For example, this documentprovides methods and materials for using the ratio of tumor-infiltratingTh2 lymphoid cells to tumor-infiltrating Th1 lymphoid cells to identifybladder cancer patients (e.g., Tis bladder cancer patients) who arelikely to respond to an anti-cancer immunotherapy. Examples ofanti-cancer immunotherapy include, without limitation, BCG therapy orα-interferon therapy. As described herein, patients having an initialbladder tissue biopsy with more tumor-infiltrating Th2 lymphoid cellsthan tumor-infiltrating Th1 lymphoid cells can be identified orclassified as being likely to respond to BCG therapy. Patients having aninitial bladder tissue biopsy that does not have more tumor-infiltratingTh2 lymphoid cells than tumor-infiltrating Th1 lymphoid cells can beidentified or classified as being unlikely to respond to BCG therapy.

A ratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltratingTh1 lymphoid cells can be determined using any appropriate method ofassessing the number of Th2 lymphoid cells and Th1 lymphoid cellspresent within a tissue sample (e.g., a tumor biopsy). For example,immunohistochemistry, immunofluorescence, or FACS® analyses techniquescan be used to assess the level of tumor-infiltrating Th2 lymphoid cellsand tumor-infiltrating Th1 lymphoid cells present within a tissuesample. When using immunohistochemistry techniques, antibodies tomarkers present on Th1 or Th2 lymphoid cells can be used. For example,antibodies such as anti-GATA-3 antibodies or anti-mouse/human GATA3antibodies can be used to assess the number of Th2 lymphoid cellspresent within tissue, while antibodies such as anti-T-bet antibodies oranti-T-bet (e.g., H-210) antibodies can be used to assess the number ofTh1 lymphoid cells present within tissue.

Once the numbers of tumor-infiltrating Th1 and Th2 lymphoid cells aredetermined, they can be compared to each other or used to determine theratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1lymphoid cells. If the patient is determined to have an initial bladdertissue biopsy with more tumor-infiltrating Th2 lymphoid cells thantumor-infiltrating Th1 lymphoid cells, then that patient can beidentified or classified as being likely to respond to BCG therapy. Ifthe patient is determined to have an initial bladder tissue biopsy withless tumor-infiltrating Th2 lymphoid cells than tumor-infiltrating Th1lymphoid cells, then that patient can be identified or classified asbeing unlikely to respond to BCG therapy. In some cases, patients havinga bladder biopsy with a ratio of tumor-infiltrating Th2 lymphoid cellsto tumor-infiltrating Th1 lymphoid cells that is greater than or equalto 3 or a cut-off ratio can be identified or classified as being likelyto respond to BCG therapy, while patients having a bladder biopsy with aratio of tumor-infiltrating Th2 lymphoid cells to tumor-infiltrating Th1lymphoid cells that is less than or equal to 1.5 or a cut-off ratio canbe identified or classified as being unlikely to respond to BCG therapy.In some cases, a cut-off ratio can be 1.5, 2.0, 2.5, or 3.0. It is to beunderstood that a ratio of tumor-infiltrating Th1 lymphoid cells totumor-infiltrating Th2 lymphoid cells can be used in a similar manner asdescribed herein regarding a ratio of tumor-infiltrating Th2 lymphoidcells to tumor-infiltrating Th1 lymphoid cells by making appropriateadjustments to the calculations.

Any appropriate sample can be used when measuring the levels oftumor-infiltrating Th1 and Th2 lymphoid cells. Such samples include,without limitation, tissue biopsies (e.g., bladder tissue biopsy) andtissue biopsy sections.

This document also provides methods and materials involve in using thelevel of eosinophil infiltration and/or degranulation to identifybladder cancer patients (e.g., Tis bladder cancer patients) who arelikely to respond to an anti-cancer immunotherapy (e.g., BCG therapy).As described herein, patients having an initial bladder tissue biopsywith elevated eosinophil infiltration and/or degranulation can beidentified or classified as being likely to respond to BCG therapy.Patients having an initial bladder tissue biopsy that lacks elevatedeosinophil infiltration and/or degranulation can be identified orclassified as being unlikely to respond to BCG therapy.

The level of eosinophil infiltration and/or degranulation present withina tissue sample (e.g., an initial bladder tissue biopsy) can bedetermined using any appropriate method. For example,immunohistochemistry, immunofluorescence, or FACS® analyses techniquescan be used to assess the level of eosinophil infiltration and/ordegranulation present within a tissue sample. When usingimmunohistochemistry techniques, antibodies to eosinophils or componentsof eosinophils can be used. For example, antibodies such as anti-humaneosinophil peroxidase antibodies and anti-mouse eosinophil peroxidaseantibodies can be used to assess the number of eosinophils ordegranulating eosinophils present within tissue.

Once the numbers of eosinophils and/or degranulating eosinophils presentwithin a tissue sample are determined, they can be compared to controlsor used to calculate a score (e.g., an eosinophil activation index(EAI)). For example, an EAI can be calculated as described herein. Insuch cases, if the patient is determined to have an initial bladdertissue biopsy with an EAI greater than 4.0, 4.5, 5.0, or 5.5, then thatpatient can be identified or classified as being likely to respond toBCG therapy. If the patient is determined to have an initial bladdertissue biopsy with an EAI less than 5.5, 5.0, 4.5, or 4.0, then thatpatient can be identified or classified as being unlikely to respond toBCG therapy.

Any appropriate sample can be used when assessing eosinophils and/ordegranulating eosinophils as described herein. Such samples include,without limitation, tissue biopsies (e.g., bladder tissue biopsy) andtissue biopsy sections.

In some cases, both the ratio of tumor-infiltrating Th2 lymphoid cellsto tumor-infiltrating Th1 lymphoid cells and the level of eosinophilinfiltration and/or degranulation can be used in combination to identifybladder cancer patients (e.g., Tis bladder cancer patients) who arelikely to respond to an anti-cancer immunotherapy (e.g., BCG therapy).For example, the ratio of tumor-infiltrating Th2 lymphoid cells totumor-infiltrating Th1 lymphoid cells can be multiplied or added to anEAI score to determine a Th2 biomarker signature that can be used toidentify bladder cancer patients who are likely to respond to ananti-cancer immunotherapy (e.g., BCG therapy) as described herein.

This document also provides kits that can be used to determine thelevels of tumor-infiltrating Th1 and Th2 lymphoid cells and/or thelevels of eosinophils and/or degranulating eosinophils. Such a kit cancontain anti-GATA-3 antibodies and anti-T-bet antibodies. In some cases,a kit containing anti-GATA-3 antibodies and anti-T-bet antibodies caninclude anti-eosinophil peroxidase antibodies (e.g., anti-humaneosinophil peroxidase antibodies). In some cases, a kit provided hereincan contain a reference chart that indicates a reference level orcut-off ratio for the levels of tumor-infiltrating Th1 and Th2 lymphoidcells and/or the levels of eosinophils and/or degranulating eosinophilsthat are indicative of patient likely or unlikely to respond to ananti-cancer immunotherapy (e.g., BCG therapy) as described herein.

This document also provides methods and materials for treating bladdercancer. For example, a bladder cancer patient can be assessed asdescribed herein to determine whether the patient is likely or unlikelyto respond to anti-cancer immunotherapy (e.g., BCG therapy). If thebladder cancer patient is identified as being likely to respond toanti-cancer immunotherapy (e.g., BCG therapy), then the patient can beadministered an anti-cancer immunotherapy. For example, such a bladdercancer patient can be administered one or more (e.g., two, three, four,or more) rounds of BCG therapy. If the bladder cancer patient isidentified as being unlikely to respond to anti-cancer immunotherapy(e.g., BCG therapy), then the patient can be administered anintravesical mitomycin C or thiotepa(N,N′N′-triethylenethiophosphoramide) chemotherapy, can be subjected toradical cystectomy, or both. For example, such a bladder cancer patientcan be subjected to a radical cystectomy procedure to treat the bladdercancer. In some cases, a bladder cancer patient that is identified asbeing unlikely to respond to anti-cancer immunotherapy (e.g., BCGtherapy) as described herein can be administered an intravesicalmitomycin C or thiotepa chemotherapy, can be subjected to radicalcystectomy, or both without having previously received an anti-cancerimmunotherapy (e.g., BCG therapy) for the treatment of bladder cancer.

In some cases, a bladder cancer patient can be assessed and treated asset forth in FIG. 6 or FIG. 7.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Assessing Bladder Cancer Outcomes

A study was performed using initial biopsies of bladder cancer patientsfrom the urogenital service. Briefly, immunohistochemistry was performedusing commercially available antibodies specific for Th2 (GATA-3⁺;anti-human/mouse GATA3 (R&D Systems; Minneapolis, Minn.); and Th1(T-bet⁺ anti-T-bet (H-210): sc-21003 (Santa Cruz Biotechnology, SantaCruz, Calif.)) lymphocytes and a monoclonal antibody specific forhuman/mouse eosinophil peroxidase (EPX-mAb). See, e.g., Protheroe etal., Clin. Gastroenterol. Hepatol., 7:749-755 (2009).

Biopsies from three subgroups of patients were used: (i) ten normalcontrol subjects (i.e., patients whose initial biopsies werepathology-negative for bladder cancer; (ii) ten Tis bladder cancerpatients unresponsive to BCG immune therapy (i.e., BCG refractory(BCG⁻)); and (iii) ten Tis bladder cancer patients who displayed noevidence of tumor follow BCG immune therapy (i.e., BCG responsivepatients (BCG⁺)).

The following was performed to determine the ratio of GATA-3⁺tumor-infiltrating lymphoid cells to T-bet⁺ tumor-infiltrating lymphoidcells (i.e., G/T ratio). Briefly, tumor-infiltrating T cells weresystematically assessed in individual Tis patient biopsies bypathologists. Intra/inter-observer reliabilities were evaluated byblinded assessments of the slides by other investigators. Six serialsections of each biopsy were taken for evaluation. Sections 1 of 6 and 6of 6 were used as isotype negative controls for the immunohistochemicalstaining of the biopsy. Section 3 of 6 was stained with Hematoxylin andEosin (H&E) for evaluation to identify the maximal focus of themononuclear proinflammatory infiltration. Sections 2 of 6 and 4 of 6were subjected to immunostaining using antibodies specific for GATA-3⁺and T-bet⁺ tumor-infiltrating T cells, respectively. Ten random highpowered (400×) fields at the maximum focus of mononuclear infiltrationwere examined, and the numbers of GATA-3⁺ (G) and T-bet⁺ (T)tumor-infiltrating T cells were enumerated to define a G/T ratio foreach patient sample. Representative photomicrographs of staining foreach T cell-specific antibody are shown in FIG. 1. Evaluations of thesesections revealed that it was possible to stratify bladder cancerpatients on the basis of G/T ratio generating two subgroups, patientswith a highly Th2 polarized immune microenvironment (G/T≧3) and patientswith a Th1 polarized subgroup with a G/T ratio≦1.5. More significantly,correlation of these scores with subsequent patient outcomes followingBCG immune therapy demonstrated that patient responsiveness to BCGtreatment (i.e., tumor elimination) trended with the higher G/T ratiopatients (FIG. 2).

EPX-mAb based immunohistochemistry was used to evaluate eosinophilinfiltration of the bladder cancer patient biopsies as well as toquantify the level of degranulation observed in each biopsy (biopsysection 5 of 6). Representative examples of this EPX-mAb-basedimmunostaining are shown in FIG. 3. These evaluations led to thedevelopment of an eosinophil activation index (EAI) expressed as theproduct of a numerical score of eosinophil tissue infiltration (0=noeosinophils/40X hpf; 1=1-4 eosinophils/40X hpf; 2=5-10 eosinophils/40Xhpf; and 3=>10 eosinophils/40X hpf) and a numerical score assessingeosinophil degranulation (1=no evidence of degranulation;2=degranulation in <20% of the biopsy; 3=degranulation evident in 20-50%of the biopsy; and 4=degranulation evident in >50% of the biopsy).Eosinophil infiltration and degranulation were unique to bladder cancerwith no evidence of either event in control (i.e., non-cancerous)bladder tissue. In addition, the initial biopsies of BCG responsivepatients (BCG⁺) displayed a higher EAI relative to BCG refractorypatients (BCG⁻) (FIG. 4).

The product of the G/T ratio and the EAI score from each patient (i.e.,G/T ratio*EAI score resulted in a Th2 biomarker signature that easilystratified subjects into BCG responsiveness and unresponsive subgroups(FIG. 5).

These results demonstrate that G/T ratios, eosinophil infiltrationand/or degranulation, or both G/T ratios and eosinophil infiltrationand/or degranulation can be used to assess bladder cancer biopsies(e.g., initial Tis bladder tumor biopsies) to determine whether or notthe patient will be responsiveness to BCG immune therapy.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for treating bladder cancer, whereinsaid method comprises: (a) detecting the presence, in bladder cancertissue of a bladder cancer patient not previously receiving BCG therapyto treat said bladder cancer, of a ratio of tumor-infiltrating Th2lymphoid cells to tumor-infiltrating Th1 lymphoid cells indicative of adecreased likelihood of responding to an anti-cancer immunotherapy, and(b) administering, to treat said bladder cancer, intravesical mitomycinC or thiotepa chemotherapy to said patient who did not previouslyreceive BCG therapy to treat said bladder cancer.
 2. The method of claim1, wherein said patient is a human patient.
 3. The method of claim 1,wherein said ratio is a ratio of GATA-3⁺ to T-bet⁺ tumor-infiltrating Tcells.
 4. The method of claim 1, wherein said method comprises detectingthe presence, in bladder cancer tissue of a bladder cancer patient, of alevel of eosinophil infiltration or degranulation indicative of adecreased likelihood of responding to said anti-cancer immunotherapy. 5.The method of claim 1, wherein said anti-cancer immunotherapy is BCGtherapy.
 6. The method of claim 1, wherein said intravesical mitomycin Cor thiotepa chemotherapy is administered to said patient before saidpatient receives BCG therapy.